PROJECT SUMMARY Tissue-engineered vascular grafts (TEVGs) constructed with human vascular smooth muscle cells (VSMCs) provide a valuable tool for addressing rampant cardiovascular disease. Due to their self-renewal and patient- specificity, induced pluripotent stem cell (iPSCs) from conversion of a person?s own somatic cells by ectopic expression of stem cell factors are the preferred cell-source for TEVGs. Before clinical use, studies on a large animal model simulating patient?s iPSC-based TEVG implantation are important to evaluate safety and efficacy of iPSC-based TEVG. Pigs are an excellent model due to their similarity to human physiology, affordability, and lack of ethical issues, compared to non-human primate models. However, genuine pig iPSCs (piPSCs) free of ectopic reprogramming factors have not been developed. My group recently derived piPSCs from inbred Massachusetts General Hospital (MGH) miniature swine, whose pluripotency is dependent on the doxycycline (DOX)-inducible expression of reprogramming factors. My preliminary data suggest there is an enhancement of pig pluripotency when epigenetic or biomechanical stiffness signaling is modified. With true, inbred piPSCs, functional VSMCs could be derived that would enable us to produce piPSC-based TEVGs for testing in multiple inbred pigs, since inbreeding overcomes immune-incompatibility issue between individuals. With this in mind, my overarching hypothesis is that pig TEVGs can be derived from true piPSCs and maintain suitable mechanical properties for implantation. In Aim 1, I will reprogram pig cells into true iPSCs by modifying their heterochromatin state and biomechanical signaling through addition of factors demonstrated to be able to overcome resistance to reprogramming in publications and in my own data. I will gain mechanistic insight into pig pluripotency via transcriptomic, epigenetic, and mechanical biosensing analysis of true piPSCs compared to transgene dependent piPSCs. In Aim 2, I will derive VSMCs from true piPSCs based on our previous studies and use piPSC-VSMCs to optimize the growth factors and basal media for vessel engineering, based on proliferation, marker expression, and collagen synthesis. We will generate inbred piPSC-based TEVGs in a pulsatile bioreactor system and compare collagen and elastin content and mechanical properties between pig iPSC- VSMC and primary VSMC-based TEVGs. The success of this proposal will set the stage for testing iPSC-based TEVG in a preclinical large animal model, producing essential knowledge for patient-specific, autologous vascular graft treatment of cardiovascular diseases. With ?pig to pig? preclinical evaluation of inbred iPSC-TEVG, we will obtain important knowledge from pigs, which will set the foundation for ?human to human? clinical application in the future.